To understand the structure of the soluble region of Enterovirus 68 3A protein, we construct a prokaryotic expression vector expressing the soluble region of EV-D68 3A protein, and identify the forms of expression product after purification. The EV-D68 3A(1-61) gene was amplified by PCR and then cloned into the expression vector pET-28a-His-SUMO. The recombinant plasmid was transformed into Escherichia coli BL21 induced by IPTG to express the fusion protein His-SUMO-3A(1-61). The recombinant protein was purified by Ni-NTA Agarose and cleaved by ULP Protease to remove His-SUMO tag. After that, the target protein 3A(1-61) was purified by a series of purification methods such as Ni-NTA, anion exchange chromatography and gel filtration chromato- graphy. Chemical cross-linking reaction assay was taken to determine the multiple polymerization state of the 3A soluble region. A prokaryotic expression vector pET28a-His-SUMO-3A(1-61) expressing the solution region of EV-D68 3A was successfully constructed and plenty of highly pure target proteins were obtained by multiple purification steps . The total protein amount was about 5 mg obtained from 1L Escherichia coli BL21 with purity > 95%. At the same time, those results determined the homomultimer form of soluble 3A construct. These data demonstrated that the expression and purification system of the soluble region of 3A were successfully set up and provide some basic konwledge for the research about 3A crystal structure and the development of antiviral drugs targeted at 3A to block viral replication.
Amino Acid Sequence ; Enterovirus D, Human ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics ; metabolism

Objective To evaluate the relationship between serum matrix metalloproteinase-9(MMP-9), white blood cell(WBC)count and type 2 diabetes mellitus with macroangiopathy and to investigate the mechanism of the protective effects of rusiglitazone(RSG)on blood vessel.Methods Serum MMP-9 was determined by ELISA in 30 normal controls and 80 type 2 diabetic patients(including 40 cases with macroangiopathy and 40 without maeroangiopathy).WBC count and other clinical parameters were also determined.32 type 2 diabetic patients received RSG(4rag qd)for 12 weeks.All parameters were determined after 4 weeks(17 patients)and after 12 weeks(all patients)to observe the changes in MMP-9,WBC and other parameters.Results In the diabetic patients,serum MMP-9 and WBC were markedly higher as compared with normal controls;and MMP-9 and WBC in patients with macroangiopathy[579(440-949)?g/L,(7.51?1.47)?10~9/L]were higher than those [324(275-423)?g/L,(6.22?0.79)?10~9/L]in the cases without macroangiopathy(P

Carotid Artery, Internal ; pathology ; Female ; Humans ; Middle Aged ; Posterior Cerebral Artery ; pathology

Eleven old male patients with hyperuricemia were collected ( hyperuricemia group,65-90 years old ).10 healthy middle-aged males ( middle-aged group,30-40 years old) and 10 healthy old males ( older group 60-70 years old ) with normal blood uric acid level were used as controls.All of the subjects were given low purine content diet ( 250 mg/d ) for 3 days followed by high purine content diet ( 800 mg/d ) consecutively for another three days.The samples of fasting blood and 24 h urine were collected for assay.The results showed that there were no significant changes of serum uric acid ( UA ) concentration in three groups after low purine content diet.But the levels of serum UA in three groups all increased significantly after high purine content diet,and the change was higher in hyperuricemia group than middle-aged group [ ( 507.7 ± 108.1 vs 378.9 ± 80.1 ) μmol/L,P＜0.05 ].24 h urine uric acid excretion in three groups was all significantly decreased after low purine content diet and increased after high purine content diet.After high purine content diet,24 h urine uric acid was lower in hyperuricemia group than middle-aged group [ ( 2.99 ± 1.21 vs 3.62 ± 1.02 ) mmol/24 h,P＜0.05 ].Blood urea nitrogen levels in all subjects decreased after low purine content diet and increased after high purine content diet ( P＜0.05 or P＜0.01 ).Creatinine clearance rate in hyperuricemia group was decreased after high purine content diet compared with baseline [ (75.3 ± 20.3 vs 80.7 ±20.0) ml/min ],and there were no significant changes in other groups after low and high purine content diet.24 h urine protein in hyperuricemia group was higher than middle-aged group ( P＜0.05 ),and increased after high purine content diet with significant difference ( P＜0.05 ).These results suggest that high purine content diet and decreased by renal uric acid clearance mainly contribute to hyperuricemia in old people.

China ; epidemiology ; Dust ; prevention & control ; Humans ; Silicon Dioxide ; chemistry ; Silicosis ; epidemiology ; prevention & control

Administration, Oral ; Adult ; Alanine Transaminase ; blood ; DNA, Viral ; blood ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; blood ; drug therapy ; Humans ; Interferons ; administration & dosage ; therapeutic use ; Male ; Nucleosides ; administration & dosage ; therapeutic use ; Pyrimidinones ; administration & dosage ; therapeutic use ; Retreatment ; Thymidine ; analogs & derivatives ; Treatment Outcome ; Virus Replication ; drug effects

OBJECTIVETo investigate the expression of microRNA-10a-5p (miR-10a) in laryngeal epithelial premalignant lesions (LEPL) and to analyze the correlations of its dysregulation with clinicopathologic parameters of LEPL.

METHODSAccording to the WHO classification (2005), 94 cases of LEPL were grouped into mild dysplasia (MID), moderate dysplasia (MOD), severe dysplasia (SD) and carcinoma in situ ( CIS). The expression of miR-10a in 94 cases of LEPL was detected by quantitative real-time polymerase chain reaction (qRT-PCR), and correlated with the clinical and follow-up data of all LEPL patients.

RESULTSmiR-10a was down-regulated in LEPL with increasing grade of dysplasia. There was significantly statistical difference between low-risk ( MID + MOD) and high-risk ( SD + CIS) lesion groups (P = 0.03). The linear regression analysis showed that miR-10a was correlated with grade and gender of LEPL (P < 0.05).

CONCLUSIONSDown regulation of miR-10a may be a useful molecular marker for grading of LEPL and diagnosis of early laryngeal cancer.

Biomarkers ; Carcinoma in Situ ; metabolism ; pathology ; Down-Regulation ; Humans ; Hyperplasia ; pathology ; Laryngeal Neoplasms ; metabolism ; pathology ; Larynx ; pathology ; MicroRNAs ; metabolism ; Precancerous Conditions ; metabolism ; pathology ; RNA, Neoplasm ; metabolism ; Real-Time Polymerase Chain Reaction

Objective To evaluate the value of MicroPure imaging system of ultrasonography in display of breast calcification,and explore its clinical application value.Methods One hundred and forty-one patients underwent by mammography,conventional ultrasound imaging and MicroPure imaging,the mammography results were reference standard,observing the display rate of MicroPure imaging for calcification,and the value of MicroPure imaging in the display of calcification with different shapes on mammography.As the pathologic diagnosis was golden standard,the accuracy of diagnosis of conventional ultrasound,conventional ultrasound combined with MicroPure imaging,conventional ultrasound combined with mammography were evaluated with receiver operating characteristic(ROC) curve.Results The display case of calcifications within breast masses:the specificity of MieroPure imaging was lower than conventional ultrasound(88.14％ vs 100％,P ＜ 0.05),the sensitivity and accuracy were higher than conventional ultrasound(100％ vs 62.79％,93.14％ vs 84.31 ％,P ＜0.05).The display case of microcalcifications within normal breast tissue:the specificity and accuracy of MicroPure software system were lower than conventional ultrasound(9.26％ vs 100％,43.87％ vs 65.96％,P ＜0.05),the sensitivity was higher than conventional ultrasound (100％ vs 20.00％,P ＜0.05).The punctate calcification,amorphous calcification,rough uneven,pleomorphic calcifications on mammography were all hyperechoic spots on MicroPure software system.The area under the ROC curve of the conventional ultrasound,conventional ultrasound combined with MicroPure imaging,conventional ultrasound combined with MicroPure imaging was 0.881,0.858,0.932,respectively.Conclusions MicroPure imaging has some clinical value on microcalcifications within breast masses,which can improve its sensitivity and accuracy.The specificity of MicroPure imaging diagnostic ultrasound imaging of microcalcifications were lower,and it can not distinguish between different shapes of calcifications.Compared with conventional ultrasound,MicroPure imaging system can not improve the accuracy of diagnosis of benign and malignant breast lesions.

Objective:Taking Chongyang county of Hubei Province as an example, the paper describes the in-tegration of the maternal and child health ( MCH) and family planning ( FP) service system and analyses the key ele-ments to provide reference for promoting integration. Methods: qualitative interviews and quantitative questionnaire were used to collect data. The software QSR Nvivo 8. 0 and SPSS 17. 0 were used for qualitative and quantitative data analysis. Results:Based on the correct understanding of integration, Chongyang implements the supporting policies actively, maintains the original compensation mode unchanged, follows the principle of“no reducing headcounts and no downsizing” strictly, promotes the merger of institutions rapidly, adjusts the service contents and methods reasona-bly, and integrates the maternal and child and family planning information platform. Conclusion:The correct concept of integration, appropriate staffing, and stable funding in Chongyang provide rich experiences for future research. However, there is room for improvement in staffing, incentive mechanisms, service content, and forms.

In the basis of a large amount of literatures, this article sumed up the characteristics and application of eggshell membrane.